A major area of interest in genome biology, especially in light of the determination of the complete nucleotide sequences of a number of genomes, is the targeted alteration of genome sequences. One such alteration is deletion, i.e., removal of sequences from a genome. Deletions can be as small as a single nucleotide pair, or can encompass hundreds, thousands or even millions of nucleotide pairs. The ability to reproducibly induce targeted deletions is useful in the identification of gene function (e.g., by gene “knock-out” studies) and can also be useful for inactivating genes (e.g., viral receptors) whose function is required for pathological processes.
Induction of small deletions by targeted cleavage of chromosomal DNA using zinc finger/nuclease fusion proteins (ZFNs) has been described. See, for example, WO 03/87341 and U.S. Patent Publication No. 2005/0064474. In brief, when a ZFN dimer (or any site-specific nuclease) is expressed within a cell, the process of targeted cleavage, followed by error-prone repair, can lead to generation of deletions, most of which are of fewer than about 20 bases, at or near the site of nuclease cleavage.
The process of ZFN-mediated mutagenesis as currently implemented using a single ZFN dimer has a number of limitations. First, the sizes of the deletions introduced by this method are generally quite small. Although deletions in excess of 100 bp are occasionally seen, the vast majority of deletions (probably more than 90%) are of fewer than about 20 bp. Therefore the method is unsuitable for generating large deletions at high efficiency. The ability to generate large deletions at high frequency would be required if, for example, it were necessary to eliminate entire regulatory region of a gene.
A second shortcoming of existing methods for ZFN-mediated mutagenesis is that the heterogeneity of the deletions, coupled with their small sizes, makes it extremely difficult to monitor or quantify the mutagenesis process using conventional approaches such as PCR. By contrast, larger deletions are much more readily detected and quantified in a background of excess unmodified gene sequence using a standard method such as PCR followed by agarose gel electrophoresis.
Thus, methods for reproducibly obtaining large deletions of chromosomal sequence at high frequency would be useful in a number of areas of genome biology.